I’ m performing dual luciferase assay experiments using 1 µg of pgl4.10[luc2] and 100ng of pgl4.74[RLuc] as reporter control ( firefly: renilla 10: 1). My transfection efficiency on HeLa cells seems to be too high with a signal luminescence greater than 106 , 107 . I was wondering if someone has got the same problem and could suggest me how to resolve it.
Just dilute your sample before reading, that is we always do when the RLUs are too high.
Thank you jonathan, I'll try as soon as possible. Do you usually use PLB or water to dilute samples?
Like Jonathan says dilute your lysate or us lesser material. Instead of 20 µl recommend by Promega in experiments with extrem high signals we used between 2-5 µl lysate. You can even downscale the whole setting to save reagents but this dependents on your luminometer and the injectors.
So you have used directly 2-5 µl of lysate without diluting with water? Therefore you have reduced the amount of buffers? For 5 µl lysate, 25µl of LARII and Stop&Glo? Thank you very much for your help
First we used 2-5 µl with 100 µl LARII and Stop& Glo and then changed to 50 µl LARII and Stop&Glo because with 25 µl the variance between the duplicate extended to much.
Hi, I know this was 3 years ago, but Lucia Brandimarte did you manage to get good values for your experiment? I'm asking because since using our new LarII and S&G from our new kit, our Luminescene signals have been quite high, up to about 10^6.
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